Absorbable topical hemostat

ABSTRACT

An absorbable spun, cotton-like topical hemostat containing fibers entangled with each other and being made of atelcollagen obtained by reconstituting solubilized collagen. Each of the fibers has a diameter of 10 to 70 μm and a length of 3 to 70 mn. At least a part of the collagen molecules constituting the fibers are crosslinked by heat at a temperature of 50° to 200° C. The hemostat is swellable upon contact with blood. In use, the hemostat readily adapts to the shape of the hemorrhagic site, has an adhesiveness to a bleeding surface and provides an effective suppression of hemorrhage.

TECHNICAL FIELD

This invention relates to an absorbable topical hemostat comprising acotton-like product of collagen fibers to be used in the field ofsurgery, particularly to an absorbable topical hemostat which can beapplied rapidly and effectively to hemorrhage from parenchymatousinternal organs and capillary hemorrhage.

BACKGROUND ART

As a hemostatic method during a surgical operation, there may bementioned a compression method, a ligation method, an electrocoagulationmethod, application of a physiologically active substance such asthrombin, fibrin paste, etc. and others. To an arterial hemorrhage withclear hemorrhagic sites, a ligation method or an electrocoagulationmethod has generally been applied. In the case of a venous hemorrhage,hemostasis is easily and sufficiently obtained by compression. However,these hemostatic methods are sometimes ineffectual to control ahemorrhage from parenchymatous internal organs and capillary hemorrhage,and when there is a tendency of hemorrhage in hepatic insufficiency orin the field of cardiovascular surgery, hemostasis is particularlydifficult. In such a case, an absorbable topical hemostat whichaccelerates a blood coagulation reaction only by contacting it with ableeding surface, whereby thrombus is formed rapidly to inhibithemorrhage, is effective since not only is operation time shortened, butalso postoperative rehemorrhage is prevented, to contribute to safepostoperative control. For this purpose, various topical hemostats usingoxidized cellulose as a material have been developed and appliedclinically. They have advantages that they are inexpensive and haveexcellent handling property, but they have disadvantages that since theydo not use a material derived from an animal body, absorption into aliving body is slow, and a strong hemolysis reaction and a reactionrejecting a foreign body are caused. In recent years, topical hemostatsusing collagen which is protein derived from an animal body havefrequently been applied clinically since the collagen has lowantigenicity and is absorbed into a living body safely to minimize anallergic reaction and a reaction rejecting a foreign body and furtherthe collagen itself also has physiological functions (coagulation ofplatelets by adhesion, acceleration of a blood coagulation system by theplatelet factor III released from coagulated platelets and formation ofthrombus) thus, its hemostatic effect is high.

Topical hemostats made of collagen which have been put to practical useat present include those in which microfibrils of collagen are madeflaky and those in which a collagen sponge is made plate-shaped. As tothe former, telopeptide which is a determinant of antigenicity ofcollagen remains to exhibit antigenicity in a living body, so that theyare required to be removed after use, they are flaky, so that they areflowed by blood to be scattered, whereby a large hemostatic effectcannot be expected, and they are easily charged with static electricity,so that there is a drawback in handling that they are easily attached tohands and tweezers during use. On the other hand, as to the latter, someof them comprise atelocollagen in which telopeptide has been removed,but they are plate-shaped, so that adhesiveness to a wound surface witha complicated shape is not sufficient and also astriction cannot becarried out, whereby a large hemostatic effect cannot be expectedsimilarly as in the former.

As a material which can solve the above drawbacks, cotton-like topicalhemostats comprising atelocollagen have been published. There may bementioned, for example, a material obtained by spinning atelocollagenderived from porcine skin, drying it and forming it into a cotton-likeproduct (Shimizu et al., Artificial Internal Organs 19 (3), 1235(1990)), a material obtained by treating spun collagen with acrosslinking agent, washing it and lyophilizing it to be formed into acotton-like product with a surface on which fine crack-like fissures aremade (Japanese Provisional Patent Publication No. 61862/1992), etc.Since the former uses soluble atelocollagen as such, when it is used forhemostasis, it absorbs blood to significantly lower fiber strength,whereby it is difficult to inhibit blood flow and therefore a sufficienthemostatic effect cannot be expected. On the other hand, in the latter,there are problems that since crosslinking treatment with a crosslinkingagent is carried out prior to lyophilization, a hemostatic effectpossessed by collagen is impaired, and from the point of safety, awashing operation for completely removing an unreacted crosslinkingagent is required.

DISCLOSURE OF INVENTION

An object of the present invention is to provide a hemostat in which thetasks of the conventional hemostats made of collagen as described abovecan be solved, i.e., to provide a topical hemostat made of collagen,which has sufficient hemostatic ability, is degraded in and absorbedinto a living body safely and rapidly after use, and also has goodhandling property.

As a result of investigation for such an object, the present inventionhas been made.

That is, the present invention is a cotton-like product comprisingfibers of atelocollagen obtained by reconstituting solubilized collagen,wherein each of said fibers has a diameter of 10 to 70 μm and a fiberlength of 3 to 70 mm, and at least a part of collagen moleculesconstituting said fibers are crosslinked by heat.

In the present invention, the solubilized collagen refers to collagensubjected to solubilization by carrying out solubilization treatmentwith protease or a solubilization treatment with alkali and alsosubjected to an operation of removing telopeptide which is a determinantof antigenicity of collagen. The source of the collagen to be used as amaterial of the present invention is not particularly limited, butcollagen obtained from skin, bone, cartilage, tendon, internal organs,etc. of a mammal (e.g. a human, a cattle, a swine, a rabbit, a sheep, amouse, etc.) is generally used. Also, collagen-like protein obtainedfrom birds, fish or the like can be used.

In general, collagen subjected to solubilization treatment with proteasehas a higher activity to platelets as compared with one subjected tosolubilization treatment with alkali, so that it is preferred to usecollagen subjected to solubilization treatment with protease.

Hemostasis by the collagen hemostat is carried out by physicallysuppressing sprung blood in the first place and then exhibiting ahemostatic effect of collagen itself. Therefore, in order to make asufficient hemostatic effect, the strength of fibers constituting thehemostat is an important factor. The strength of the fibers is larger asthe diameter of the constituting fibers is larger. However, when thediameter thereof is too large, the fibers become rigid, so that shapeprocessing is difficult. Thus, the diameter may be 10 to 70 μm,preferably 15 to 45 μm.

The length of the fibers can be any desired length since said fibers areobtained by reconstituting solubilized collagen. However, when thelength thereof is too long, it is difficult to disperse the fiberssufficiently when said fibers are formed into a cotton-like product, andwhen the length thereof is too short, the fibers are hardly entangledwith each other, so that said fibers are scattered in application of thehemostat and strength resisting blood pressure cannot be obtained. Thus,the length may be 3 to 70 mm, preferably 5 to 50 mm.

The absorbable topical hemostat of the present invention is swollen byabsorbing blood to increase the whole volume thereof. As a result, anastriction effect on peripheral tissues of applied sites is exhibited.The astriction effect is exhibited only when the hemostat has a certaindegree of strength. In the fibers obtained from the solubilizedcollagen, a swelling degree thereof is significant, and the strength islowered accompanied with swelling. Therefore, in the present invention,by crosslinking at least a part of collagen molecules constituting saidfibers by heat, suitable swellability (said fibers have a dense surfacestructure, but they absorb blood rapidly to cause volume expansion,whereby an astriction effect on bleeding sites is brought about) andstrength (an action of inhibiting blood stream completely to achievehemostasis in an early stage) as a hemostat are imparted whilemaintaining a hemostatic action inherently possessed by collagen.

In the prior art, when collagen is crosslinked, there has been used amethod of using a crosslinking agent such as aldehydes, diepoxides, etc.However, in collagen treated by a crosslinking agent, activity toplatelets of collagen is lost so that improvement of hemostatic abilitycannot be expected (reactivity to platelets of collagen is hardlydeactivated by heat treatment). Further, the method of using acrosslinking agent has many problems that the reaction cannot be stoppedat the time when crosslinking proceeds to a degree that a swellingdegree suitable as a hemostat remains, and the crosslinking agent to beused has toxicity in many cases so that a washing operation for removingunreacted crosslinking agent is required.

The crosslinking treatment by heat is preferably carried out at a rangeof 50° to 200° C. If it is lower than 50° C., crosslinking becomesinsufficient or crosslinking cannot be carried out, while if it exceeds200° C., denaturation of collagen is remarkable, whereby swellabilityand physiological activity are lost. When both factors of production andquality are considered, it is more preferably 60° to 180° C.,particularly preferably 95° to 150° C.

As another physical property to be possessed by the absorbable topicalhemostat, a specific volume of the cotton-like product of collagenfibers is also important in the points of handling property of thehemostat and adhesiveness to wound sites. The specific volume of thefibers is preferably higher from easiness of shape processing inapplication (the hemostatic effect can be exhibited maximally bychanging the shape of the hemostat in accordance with wound sites havingvarious shapes and uniformly adhering it thereto). However, there is alimitation thereof in order to suitably entangle the fibers with eachother, and if it is too high, the fibers are scattered or gaps areformed in application, whereby complete hemostasis is difficult. Thus,the specific volume may be 20 to 80 cm³ /g, preferably about 40 to 70cm³ /g.

Here, the specific volume is determined as described below (hereinafter,description is made with the cotton-like product of collagen fibersbeing a standard, but the flake-like product is also measured in thesame manner).

1 Under standard conditions (20° C., 65% RH), about 1 g of thecotton-like product of collagen fibers obtained is taken and weighedcorrectly (measured weight: Wg).

Under the standard conditions, a transparent plastic tube having aninner diameter of 35 mm is uniformly filled with the product.

Next, a flat disc having a diameter of 30 mm and a weight of 5.0 g isplaced on the cotton-like product prepared in

, and 50 g of a weight is further placed thereon for 30 seconds.Thereafter, said weight is removed, and the remainder is left to standfor 30 seconds. This operation is repeated three times, and then theheight of the filled cotton-like product is measured at three positionsin the peripheral direction (the average value is defined as H mm).

The specific volumes of three samples are determined according to thefollowing equation, and an average value thereof is used.

Specific volume (cm³ /g)=((35/20)² ×π×H/10)/W

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a microphotograph (magnification: 1,000) showing a shape of asurface of atelocollagen fiber constituting the absorbable topicalhemostat of the present invention.

FIG. 2 is a microphotograph (magnification: 7,000) in which said surfaceis further enlarged.

BEST MODE FOR CARRYING OUT THE INVENTION Example 1

By treating insoluble collagen obtained from fresh cattle skin withprotease (pepsin, hereinafter the same), telopeptide was digested and asolution of solubilized atelocollagen was obtained. Next, this solutionwas dissolved in an aqueous hydrochloric acid adjusted to pH 2 (collagenconcentration: 6%), and atelocollagen fibers were obtained by a wetspinning method using 20% aqueous ammonium sulfate as a coagulatingagent. The atelocollagen fibers obtained were desalted and dehydratedwith methanol, dried and then cut to have a length of 50 mm. The fiberswere dispersed by air blow (at a wind speed of 30 m/s for 10 minutes)and then subjected to heat treatment at 105° C. (for 3 hours) to obtaina cotton-like product of the collagen fibers. The physical propertyvalues of the cotton-like product obtained were a fiber length: 5 to 50mm, a fiber diameter: 15 to 45 μm and a specific volume: 60 cm³ /g. Theshape of the fiber surface of the cotton-like product obtained is shownin FIG. 1 and FIG. 2 (which are the results of observation by a scanningtype electron microscope manufactured by Nihon Denshi Co.: ModelJSM-804).

Example 2

A cotton-like product of collagen fibers was obtained in the same manneras in Example 1 except for carrying out the heat treatment at 60° C.(for 24 hours). The physical property values of the cotton-like productobtained were the same as those of Example 1.

Example 3

A cotton-like product of collagen fibers was obtained in the same manneras in Example 1 except for carrying out the heat treatment at 180° C.(for 10 minutes). The physical property values of the cotton-likeproduct obtained were the same as those of Example 1.

Example 4

A cotton-like product of collagen fibers was obtained in the same manneras in Example 1 except for not carrying out the dispersion treatment ofthe fibers by air blow. The physical property values of the cotton-likeproduct obtained were a fiber length: 30 to 50 mm, a fiber diameter: 15to 45 μm and a specific volume: 28 cm³ /g.

Example 5

A cotton-like product of collagen fibers was obtained in the same manneras in Example 1 except for changing the conditions of the dispersiontreatment of the fibers by air blow to a wind speed of 40 m/s for 60minutes. The physical property values of the cotton-like productobtained were a fiber length: 5 to 50 mm, a fiber diameter: 15 to 45 μmand a specific volume: 78 cm³ /g.

Example 6

A cotton-like product of collagen fibers was obtained in the same manneras in Example 1 except for carrying out alkali solubilization treatment(using a mixed solution of sodium sulfate, sodium hydroxide andmonomethylamine) in place of the enzyme treatment of insoluble collagen.The physical property values of the cotton-like product obtained werethe same as those of Example 1.

Example 7

A cotton-like product of collagen fibers was obtained in the same manneras in Example 1 except for using fresh porcine skin in place of freshcattle skin. The physical property values of the cotton-like productobtained were the same as those of Example 1.

Comparative Example 1

A cotton-like product of collagen fibers was obtained in the same manneras in Example 1 except for not carrying out the heat treatment. Thephysical property values of the cotton-like product obtained were thesame as those of Example 1.

Comparative Example 2

A cotton-like product of collagen fibers was obtained in the same manneras in Example 1 except for carrying out alkali solubilization treatment(using a mixed solution of sodium sulfate, sodium hydroxide andmonomethylamine) in place of the enzyme treatment of insoluble collagenand not carrying out the heat treatment. The physical property values ofthe cotton-like product obtained were the same as those of Example 1.

Comparative Example 3

A cotton-like product of collagen fibers was obtained in the same manneras in Example 1 except for carrying out treatment with glutaraldehyde inplace of the heat treatment. The physical property values of thecotton-like product obtained were the same as those of Example 1.

Comparative Example 4

A cotton-like product of collagen fibers was obtained in the same manneras in Example 1 except for changing the spinning conditions. Thephysical properties of the cotton-like product obtained were a fiberlength: 5 to 50 mm, a fiber diameter: 7 to 9 μm and a specific volume:65 cm³ /g.

Comparative Example 5

A cotton-like product of collagen fibers was obtained in the same manneras in Example 1 except for changing the spinning conditions. Thephysical properties of the cotton-like product obtained were a fiberlength: 5 to 50 mm, a fiber diameter: 80 to 100 μm and a specificvolume: 35 cm³ /g.

Comparative Example 6

A flake-like product of collagen fibers was obtained in the same manneras in Example 1 except for changing the cut length after drying to 3 mm.The physical property values of the flake-like product obtained were afiber length: 0.5 to 1 mm, a fiber diameter: 15 to 45 μm and a specificvolume: 18 cm³ /g.

Comparative Example 7

By treating insoluble collagen obtained from fresh cattle skin withprotease, telopeptide was digested and a solution of solubilizedatelocollagen was obtained. Next, this solution was dissolved in anaqueous hydrochloric acid adjusted to pH 2 (collagen concentration: 3%),and collagen fibers were obtained by a wet spinning method using asaturated solution of sodium sulfate as a coagulating agent. Next, thesefibers were treated with a glutaraldehyde solution and then washed withwater to remove inorganic salts and unreacted glutaraldehyde. Next, thefibers were cut to have a length of 50 mm and lyophilized to obtain acotton-like product of the collagen fibers. The physical properties ofthe cotton-like product obtained were a fiber length: 40 to 50 mm, afiber diameter: 10 to 30 μm and a specific volume: 17 cm³ /g.

Experiment

Mongrel adult dogs to which 100 u/kg of heparin had been systemicallyadministered before initiation of the experiment were subjected tolaparotomy under general anesthesia, and only spleen integument with anexact size of 1 cm×1 cm was peeled off by a surgical knife. Next, each0.1 g of the collagen cotton-like products and flake-like productsobtained in the respective Examples and Comparative examples describedabove were put on the above peeled surfaces and compressed for 30seconds, and bleeding amounts were measured at intervals of 1 minute.Dried gauze was put on a bleeding surface for 1 minute, and the bleedingamount was determined by a radius of a circle made by blood absorbed bythe gauze. This determination is set forth as an index which ishereinafter referred to as Bleeding Degree: BD. An initial value of BDis defined as BD_(t=0), BD with each lapse of time is defined asBD_(t=t), and a value determined according to the following equation isdefined as a hemostatic rate: HR. At the time when no blood permeatedinto the gauze, hemostasis was regarded to be completed.

    HR (%)=(1-(BD.sub.t=t /BD.sub.t=0))×100

Data obtained when the respective 10 samples were examined are shown inTable 1. In said table, also shown are results of measuring a timerequired for processing 0.1 g of the collagen cotton-like products andthe flake-like products obtained in the respective Examples andComparative examples described above in accordance with the shape of thepeeled surfaces and applying them to the peeled surfaces (referred to asa reprocessing time).

From the results, it is apparent that the hemostatic collagencotton-like products of the present invention have excellent hemostaticability and excellent handling property. In experiment examples of thisexperiment, in which hemostasis was not completed in an early stage ofhemostasis (in 2 minutes), exuded blood was sprung out by partiallypushing through the hemostat (because the fibers constituting thehemostat could not maintain strength enough to resist blood pressure),whereby hemostasis was difficult. That is, it is extremely importantthat hemostasis is completed in 2 minutes.

                  TABLE 1                                                         ______________________________________                                        HR (%)                     Reprocess-                                         After      After    After  After  After                                                                              ing time                               1 min      2 min    3 min  4 min  5 min                                                                              (sec)                                  ______________________________________                                        Example 1                                                                             100    ←   ←                                                                             ← ←                                                                             2                                    Example 2                                                                             51     100      ←                                                                             ← ←                                                                             2                                    Example 3                                                                             53     100      ←                                                                             ← ←                                                                             2                                    Example 4                                                                             75     100      ←                                                                             ← ←                                                                             5                                    Example 5                                                                             69     100      ←                                                                             ← ←                                                                             3                                    Example 6                                                                             88     100      ←                                                                             ← ←                                                                             2                                    Example 7                                                                             100    ←   ←                                                                             ← ←                                                                             2                                    Comparative                                                                           35     95       63   100    ←                                                                             2                                    example 1                                                                     Comparative                                                                           18     24       ←                                                                             ← ←                                                                             2                                    example 2                                                                     Comparative                                                                           59     67       ←                                                                             ← ←                                                                             3                                    example 3                                                                     Comparative                                                                           31     40       59   ← ←                                                                             3                                    example 4                                                                     Comparative                                                                           36     54       ←                                                                             ← ←                                                                             6                                    example 5                                                                     Comparative                                                                           56     86       ←                                                                             ← ←                                                                             impossible                           example 6                                                                     Comparative                                                                           38     46       62   ← ←                                                                             6                                    example 7                                                                     ______________________________________                                    

INDUSTRIAL APPLICABILITY

The cotton-like product of collagen fibers according to the presentinvention is a gathering of fibers which are obtained by reconstitutingsolubilized collagen and have suitable length, diameter and specificvolume. Since at least a part of collagen molecules constituting saidfibers are crosslinked by heat, a suppression effect on hemorrhage whichis the first stage of hemostasis is improved, and sufficient hemostaticability is exhibited without impairing a hemostatic activity inherentlypossessed by collagen. Also, since it has a suitable specific volume, itcan be easily processed in accordance with a shape of a hemorrhagicsite, it can be used for hemostasis rapidly, and also adhesiveness to ableeding surface is good, whereby a hemostatic effect is high.

We claim:
 1. An absorbable spun, cotton-like topical hemostat comprisingfibers entangled with each other and being made of atelcollagen obtainedby reconstituting solubilized collagen, wherein each of said fibers hasa diameter of 10 to 70 μm and a fiber length of 3 to 70 mm, and at leasta part of collagen molecules constituting said fibers are crosslinked byheat at a temperature of 50° to 200° C., the hemostat being swellableupon contact with blood, the hemostat having a shape which is changeableto conform to a wound site, and the hemostat having a specific volume of20 to 80 cm³ /g.
 2. The absorbable topical hemostat according to claim1, wherein the diameter of the fibers is 15 to 45 μm.
 3. The absorbabletopical hemostat according to claim 1, wherein the length of the fibersis 5 to 50 mm.
 4. The absorbable topical hemostat according to claim 2,wherein the length of the fibers is 5 to 50 mm.
 5. The absorbabletopical hemostat according to claim 4, wherein the crosslinking iscarried out at a temperature of 60° to 180° C.
 6. The absorbable topicalhemostat according to claim 4, wherein the crosslinking is carried outat a temperature of 95° to 150° C.
 7. The absorbable topical hemostataccording to claim 1, wherein the specific volume is 40 to 70 cm³ /g. 8.The absorbable topical hemostat according to claim 4, wherein thespecific volume is 40 to 70 cm³ /g.
 9. The absorbable topical hemostataccording to claim 1, wherein the hemostat is made by contactinginsoluble collagen obtained from the skin, bone, cartilage, tendon or aninternal organ of a mammal with a protease to digest telopeptide andform a solution of solubilized atelocollagen, dissolving the solution inaqueous hydrochloric acid to obtain atelocollagen fibers, wet spinningthe atelocollagen fibers with aqueous ammonium sulfate, desalting anddehydrating the spun atelocollagen fibers with methanol, drying andcutting the spun atelocollagen fibers to a desired length, air blowingthe fibers and subjecting the fibers to a heat treatment at atemperature of 50° to 200° C.